Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

The ionotropic glutamate receptors (GluR) are the primary mediators of exci tatory synaptic transmission in the brain. GluR agonist binding has been lo calized to an extracellular domain whose core is homologous to the bacteria l periplasmic binding proteins (PBP). We have established routine, baculovi rus-mediated expression of a complete ligand-binding domain construct at th e 10-L scale, yielding 10-40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connec t the domain core to the transmembrane domains of the channel and to the N- terminal 'X'-domain. These linker peptides have been implicated in modulati ng channel physiology. Such extended constructs have proven difficult to ex press in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the doma in involves a substantial heat capacity change and that at physiological te mperatures, the reaction is both entropically and enthalpically favorable.